Structural assignments to the Mycoplasma genitalium proteins show extensive gene duplications and domain rearrangements

The parasitic bacterium Mycoplasma genitalium has a small, reduced genome with close to a basic set of genes. As a first step toward determining the families of protein domains that form the products of these genes, we have used the multiple sequence programs psi-blast and geanfammer to match the sequences of the 467 gene products of M. genitalium to the sequences of the domains that form proteins of known structure [Protein Data Bank (PDB) sequences]. PDB sequences (274) match all of 106 M. genitalium sequences and some parts of another 85; thus, 41% of its total sequences are matched in all or part. The evolutionary relationships of the PDB domains that match M. genitalium are described in the structural classification of proteins (SCOP) database. Using this information, we show that the domains in the matched M. genitalium sequences come from 114 superfamilies and that 58% of them have arisen by gene duplication. This level of duplication is more than twice that found by using pairwise sequence comparisons. The PDB domain matches also describe the domain structure of the matched sequences: just over a quarter contain one domain and the rest have combinations of two or more domains.

Modeling of domain growth activity in polycrystalline ferrites.

"Supported in part by Contract No. U.S. AEC AT(11-1)-1018."

Design of high quality doped CeO2 solid electrolytes with nanohetero structure

Doped ceria (CeO2) compounds are fluorite related oxides which show oxide ionic conductivity higher than yttria-stabilized zirconia in oxidizing atmosphere. As a consequence of this, a considerable interest has been shown in application of these materials for low (400-650 degrees C) temperature operation of solid oxide fuel cells (SOFCs). In this paper, our experimental data about the influence of microstructure at the atomic level on electrochemical properties were reviewed in order to develop high quality doped CeO2 electrolytes in fuel cell applications. Using this data in the present paper, our original idea for a design of nanodomain structure in doped CeO2 electrolytes was suggested. The nanosized powders and dense sintered bodies of M doped CeO2 (M:Sm,Gd,La,Y,Yb, and Dy) compounds were fabricated. Also nanostiructural features in these specimens were introduced for conclusion of relationship between electrolytic properties and domain structure in doped CeO2. It is essential that the electrolytic properties in doped CeO2 solid electrolytes reflect in changes of microstructure even down to the atomic scale. Accordingly, a combined approach of nanostructure fabrication, electrical measurement and structure characterization was required to develop superior quality doped CeO2 electrolytes in the fuel cells.

Structure and lipid interactions of membrane-associated glycosyltransferases : Cationic patches and anionic lipids regulate biomembrane binding of both GT-A and GT-B enzymes

This thesis concerns work on structure and membrane interactions of enzymes involved in lipid synthesis, biomembrane and cell wall regulation and cell defense processes. These proteins, known as glycosyltransferases (GTs), are involved in the transfer of sugar moieties from nucleotide sugars to lipids or chitin polymers. Glycosyltransferases from three types of organisms have been investigated; one is responsible for vital lipid synthesis in Arabidopsis thaliana (atDGD2) and adjusts the lipid content in biomembranes if the plant experiences stressful growth conditions. This enzyme shares many structural features with another GT found in gram-negative bacteria (WaaG). WaaG is however continuously active and involved in synthesis of the protective lipopolysaccharide layer in the cell walls of Escherichia coli. The third type of enzymes investigated here are chitin synthases (ChS) coupled to filamentous growth in the oomycete Saprolegnia monoica. I have investigated two ChS-derived MIT domains that may be involved in membrane interactions within the endosomal pathway. From analysis of the three-dimensional structure and the amino-acid sequence, some important regions of these very large proteins were selected for in vitro studies. By the use of an array of biophysical methods (e.g. Nuclear Magnetic Resonance, Fluorescence and Circular Dichroism spectroscopy) and directed sequence analyses it was possible to shed light on some important details regarding the structure and membrane-interacting properties of the GTs. The importance of basic amino-acid residues and hydrophobic anchoring segments, both generally and for the abovementioned proteins specifically, is discussed. Also, the topology and amino-acid sequence of GT-B enzymes of the GT4 family are analyzed with emphasis on their biomembrane association modes. The results presented herein regarding the structural and lipid-interacting properties of GTs aid in the general understanding of glycosyltransferase activity. Since GTs are involved in a high number of biochemical processes in vivo it is of outmost importance to understand the underlying processes responsible for their activity, structure and interaction events. The results are likely to be useful for many applications and future experimental design within life sciences and biomedicine.

Validation of the WHOQOL-BREF among women following childbirth

Background: There is an increasing interest in measuring quality of life (QOL) in clinical settings and in clinical trials. None of the commonly used QOL instrument have been validated for use postnatally. Aim: To assess the psychometric properties of the 26-item WHOQOL-BREF among women following childbirth. Methods: Using a prospective cohort design we recruited 320 women within the first few days of childbirth. At six weeks postpartum, participants were asked to complete the WHOQOL-BREF, the Edinburgh Postnatal Depression Index and the Australian Unity Wellbeing Index. Validation of the WHOQOL-BREF included an analysis of internal consistency, discriminate validity, convergent validity and an examination of the domain structure. Results: 221 (69.1%) women returned their six-week questionnaire. All domains of the WHOQOL-BREF met reliability standards (alpha coefficient exceeding 0.70). The questionnaire discriminated well between known groups (depressed and non-depressed women. P = <0.000) and demonstrated satisfactory correlations with the Australian Unity Wellbeing index (r = >0.45). The domain structure of the WHOQOL-BREF was also valid in this population of new mothers, with moderate to high correlation between individual items and the domain structure to which the items were originally assigned. Conclusion: The WHOQOL-BRF is well-accepted and valid instrument in this population and may be used in postnatal clinical settings or for assessing intervention effects in research studies.

Deriving a preference-based measure for cancer using the EORTC QLQ-C30 : a confirmatory versus exploratory approach

Background: To derive preference-based measures from various condition-specific descriptive health-related quality of life (HRQOL) measures. A general 2-stage method is evolved: 1) an item from each domain of the HRQOL measure is selected to form a health state classification system (HSCS); 2) a sample of health states is valued and an algorithm derived for estimating the utility of all possible health states. The aim of this analysis was to determine whether confirmatory or exploratory factor analysis (CFA, EFA) should be used to derive a cancer-specific utility measure from the EORTC QLQ-C30. Methods: Data were collected with the QLQ-C30v3 from 356 patients receiving palliative radiotherapy for recurrent or metastatic cancer (various primary sites). The dimensional structure of the QLQ-C30 was tested with EFA and CFA, the latter based on a conceptual model (the established domain structure of the QLQ-C30: physical, role, emotional, social and cognitive functioning, plus several symptoms) and clinical considerations (views of both patients and clinicians about issues relevant to HRQOL in cancer). The dimensions determined by each method were then subjected to item response theory, including Rasch analysis. Results: CFA results generally supported the proposed conceptual model, with residual correlations requiring only minor adjustments (namely, introduction of two cross-loadings) to improve model fit (increment χ2(2) = 77.78, p < .001). Although EFA revealed a structure similar to the CFA, some items had loadings that were difficult to interpret. Further assessment of dimensionality with Rasch analysis aligned the EFA dimensions more closely with the CFA dimensions. Three items exhibited floor effects (>75% observation at lowest score), 6 exhibited misfit to the Rasch model (fit residual > 2.5), none exhibited disordered item response thresholds, 4 exhibited DIF by gender or cancer site. Upon inspection of the remaining items, three were considered relatively less clinically important than the remaining nine. Conclusions: CFA appears more appropriate than EFA, given the well-established structure of the QLQ-C30 and its clinical relevance. Further, the confirmatory approach produced more interpretable results than the exploratory approach. Other aspects of the general method remain largely the same. The revised method will be applied to a large number of data sets as part of the international and interdisciplinary project to develop a multi-attribute utility instrument for cancer (MAUCa).

Deriving a preference-based utility measure for cancer patients from the European Organisation for the Research and Treatment of Cancer's Quality of Life Questionnaire C30 : a confirmatory versus exploratory approach

Background Multi attribute utility instruments (MAUIs) are preference-based measures that comprise a health state classification system (HSCS) and a scoring algorithm that assigns a utility value to each health state in the HSCS. When developing a MAUI from a health-related quality of life (HRQOL) questionnaire, first a HSCS must be derived. This typically involves selecting a subset of domains and items because HRQOL questionnaires typically have too many items to be amendable to the valuation task required to develop the scoring algorithm for a MAUI. Currently, exploratory factor analysis (EFA) followed by Rasch analysis is recommended for deriving a MAUI from a HRQOL measure. Aim To determine whether confirmatory factor analysis (CFA) is more appropriate and efficient than EFA to derive a HSCS from the European Organisation for the Research and Treatment of Cancer’s core HRQOL questionnaire, Quality of Life Questionnaire (QLQ-C30), given its well-established domain structure. Methods QLQ-C30 (Version 3) data were collected from 356 patients receiving palliative radiotherapy for recurrent/metastatic cancer (various primary sites). The dimensional structure of the QLQ-C30 was tested with EFA and CFA, the latter informed by the established QLQ-C30 structure and views of both patients and clinicians on which are the most relevant items. Dimensions determined by EFA or CFA were then subjected to Rasch analysis. Results CFA results generally supported the proposed QLQ-C30 structure (comparative fit index =0.99, Tucker–Lewis index =0.99, root mean square error of approximation =0.04). EFA revealed fewer factors and some items cross-loaded on multiple factors. Further assessment of dimensionality with Rasch analysis allowed better alignment of the EFA dimensions with those detected by CFA. Conclusion CFA was more appropriate and efficient than EFA in producing clinically interpretable results for the HSCS for a proposed new cancer-specific MAUI. Our findings suggest that CFA should be recommended generally when deriving a preference-based measure from a HRQOL measure that has an established domain structure.

Palladin, a novel microfilament protein

Palladin is a novel actin microfilament associated protein, which together with myotilin and myopalladin forms a novel cytoskeletal IgC2 domain protein family. Whereas the expression of myotilin and myopalladin is limited mainly to striated muscle, palladin is widely expressed in both epithelial and mesenchymal tissues, including heart and the nervous system. Palladin has a complex genetic structure and it is expressed as several different sized and structured splice variants, which also display differences in their expression pattern and interactions. In muscle cells, all the family members localize to the sarcomeric Z-disc, and in non-muscle cells palladin also localizes to the stress-fiber-dense regions, lamellipodia, podosomes and focal adhesions. A common feature of this protein family is the binding to α-actinin, but other interactions are mostly unique to each member. Palladin has been shown to interact with several proteins, including VASP, profilin, Eps8, LASP-1 and LPP. Its domain structure, lack of enzymatic activity and multiple interactions define it as a molecular scaffolding protein, which links together proteins with different functional modalities into large complexes. Palladin has an important role in cytoskeletal regulation, particularly in stress fiber formation and stabilization. This assumption is supported by several experimental results. First, over-expression of palladin in non-muscle cells results in rapid reorganization of the actin cytoskeleton and formation of thick actin bundles. Second, the knock-down of palladin with anti-sense and siRNA techniques or knock-out by genetic methods leads to defective stress fiber formation. Furthermore, palladin is usually up-regulated in situations requiring a highly organized cytoskeleton, such as differentiation of dendritic cells, trophoblasts and myofibroblasts, and activation of astrocytes during glial scar formation. The protein family members have also direct disease linkages; myotilin missense mutations are the cause of LGMD1A and myofibrillar myopathy. Palladin mutations and polymorphisms, on the other hand, have been linked to hereditary pancreatic cancer and myocardial infarction, respectively. In this study we set out to characterize human palladin. We identified several palladin isoforms, studied their tissue distribution and sub-cellular localization. Four novel interaction partners were identified; ezrin, ArgBP2, SPIN90 and Src-kinase.The previously identified interaction between palladin and α-actinin was also characterized in detail. All the identified new binding partners are actin cytoskeleton associated proteins; ezrin links the plasma membrane to the cytoskeleton, ArgBP2 and SPIN90 localize, among other structures, to the lamellipodia and in cardiomyocytes to the Z-disc. Src is a transforming tyrosine kinase, which besides its role in oncogenesis has also important cytoskeletal associations. We also studied palladin in myofibroblasts, which are specialized cells involved in diverse physiological and pathological processes, such as wound healing and tissue fibrosis. We demonstrated that palladin is up-regulated during the differentiation of myofibroblasts in an isoform specific manner, and that this up-regulation is induced by TGF-β via activation of both the SMAD and MAPK signalling cascades. In summary, the results presented here describe the initial characterization of human palladin and offer a basis for further studies.

Folding and Selective Exit of Reporter Proteins from the Yeast Endoplasmic Reticulum

The present study analyses the traffic of Hsp150 fusion proteins through the endoplasmic reticulum (ER) of yeast cells, from their post-translational translocation and folding to their exit from the ER via a selective COPI-independent pathway. The reporter proteins used in the present work are: Hsp150p, an O-glycosylated natural secretory protein of Saccharomyces cerevisiae, as well as fusion proteins consisting of a fragment of Hsp150 that facilitates in the yeast ER proper folding of heterologous proteins fused to it. It is thought that newly synthesized polypeptides are kept in an unfolded form by cytosolic chaperones to facilitate the post-translational translocation across the ER membrane. However, beta-lactamase, fused to the Hsp150 fragment, folds in the cytosol into bioactive conformation. Irreversible binding of benzylpenicillin locked beta-lactamase into a globular conformation, and prevented the translocation of the fusion protein. This indicates that under normal conditions the beta-lactamase portion unfolds for translocation. Cytosolic machinery must be responsible for the unfolding. The unfolding is a prerequisite for translocation through the Sec61 channel into the lumen of the ER, where the polypeptide is again folded into a bioactive and secretion-competent conformation. Lhs1p is a member of the Hsp70 family, which functions in the conformational repair of misfolded proteins in the yeast ER. It contains Hsp70 motifs, thus it has been thought to be an ATPase, like other Hsp70 members. In order to understand its activity, authentic Lhs1p and its recombinant forms expressed in E. coli, were purified. However, no ATPase activity of Lhs1p could be detected. Nor could physical interaction between Lhs1p and activators of the ER Hsp70 chaperone Kar2p, such as the J-domain proteins Sec63p, Scj1p, and Jem1p and the nucleotide exchange factor Sil1p, be demonstrated. The domain structure of Lhs1p was modelled, and found to consist of an ATPase-like domain, a domain resembling the peptide-binding domain (PBD) of Hsp70 proteins, and a C-terminal extension. Crosslinking experiments showed that Lhs1p and Kar2p interact. The interacting domains were the C-terminal extension of Lhs1p and the ATPase domain of Kar2p, and this interaction was independent of ATPase activity of Kar2p. A model is presented where the C-terminal part of Lhs1p forms a Bag-like 3 helices bundle that might serve in the nucleotide exchange function for Kar2p in translocation and folding of secretory proteins in the ER. Exit of secretory proteins in COPII-coated vesicles is believed to be dependent of retrograde transport from the Golgi to the ER in COPI-coated vesicles. It is thought that receptors escaping to the Golgi must be recycled back to the ER exit sites to recruit cargo proteins. We found that Hsp150 leaves the ER even in the absence of functional COPI-traffic from the Golgi to the ER. Thus, an alternative, COPI-independent ER exit pathway must exists, and Hsp150 is recruited to this route. The region containing the signature guiding Hsp150 to this alternative pathway was mapped.

Surface Proteins of Lactobacillus crispatus: Adhesive Properties and Cell Wall Anchoring

Bacterial surface-associated proteins are important in communication with the environment and bacteria-host interactions. In this thesis work, surface molecules of Lactobacillus crispatus important in host interaction were studied. The L. crispatus strains of the study were known from previous studies to be efficient in adhesion to intestinal tract and ECM. L. crispatus JCM 5810 possess an adhesive surface layer (S-layer) protein, whose functions and domain structure was characterized. We cloned two S-layer protein genes (cbsA; collagen-binding S-layer protein A and silent cbsB) and identified the protein region in CbsA important for adhesion to host tissues, for polymerization into a periodic layer as well as for attachment to the bacterial cell surface. The analysis was done by extensive mutation analysis and by testing His6-tagged fusion proteins from recombinant Escherichia coli as well as by expressing truncated CbsA peptides on the surface of Lactobacillus casei. The N-terminal region (31-274) of CbsA showed efficient and specific binding to collagens, laminin and extracellular matrix on tissue sections of chicken intestine. The N-terminal region also contained the information for formation of periodic S-layer polymer. This region is bordered at both ends by a conserved short region rich in valines, whose substitution to leucines drastically affected the periodic polymer structure. The mutated CbsA proteins that failed to form a periodic polymer, did not bind collagens, which indicates that the polymerized structure of CbsA is needed for collagen-binding ability. The C-terminal region, which is highly identical in S-layer proteins of L. crispatus, Lactobacillus acidophilus and Lactobacillus helveticus, was shown to anchor the protein to the bacterial cell wall. The C-terminal CbsA peptide specifically bound to bacterial teichoic acid and lipoteichoic acids. In conclusion, the N-terminal domain of the S-layer protein of L. crispatus is important for polymerization and adhesion to host tissues, whereas the C-terminal domain anchors the protein to bacterial cell-wall teichoic acids. Lactobacilli are fermentative organisms that effectively lower the surrounding pH. While this study was in progress, plasminogen-binding proteins enolase and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were identified in the extracellular proteome of L. crispatus ST1. In this work, the cell-wall association of enolase and GAPDH were shown to rely on pH-reversible binding to the cell-wall lipoteichoic acids. Enolase from L. crispatus was functionally compared with enolase from L. johnsonii as well as from pathogenic streptococci (Streptococcus pneumoniae, Streptococcus pyogenes) and Staphylococcus aureus. His6-enolases from commensal lactobacilli bound human plasminogen and enhanced its activation by human plasminogen activators similarly to, or even better than, the enolases from pathogens. Similarly, the His6-enolases from lactobacilli exhibited adhesive characteristics previously assigned to pathogens. The results call for more detailed analyses of the role of the host plasminogen system in bacterial pathogenesis and commensalism as well of the biological role and potential health risk of the extracellular proteome in lactobacilli.

Ferroelastic phase transition in CslO4

An optical investigation of the high-temperature structural phase transition in gel-grown single crystals of CslO4 is reported. This crystal undergoes a phase transition from the room-temperature orthorhombic phase of symmetry Pnma to a tetragonal phase at 150°C. The birefringence Δn = |na-nb| falls abruptly at Tc, indicating the first-order nature of the phase transition. Microscopic examination has revealed the existence of ferroelastic domains in the crystal. The domain structure and its dependence on temperature was studied in detail. The experimental results suggest that this crystal can be assigned to the ferroelastic Aizu species 4/mmmFmmm (p).

Switching studies and the effect of irradiation on ferroelectric properties of TAAP and DTAAP

Polarization switching processes in TAAP and DTAAP have been studied by the Merz method. The switching process in DTAAP is slower than in TAAP. The temperature dependence of switching time indicates that the crystal might contain groups of domain nuclei with different activation energies. X-ray irradiation causes an increase in the threshold field below which switching could not occur and decrease in the mobility of domain walls. Irradiation decreases the peak value of dielectric constant, Tc and increases the value of coercive field. Domain structure studies on TAAP crystals have shown that the crystals grow as both predominantly single domain and multi domains, depending on which the internal bias increases or remains unaffected upon irradiation.

Magnetic skin layer of NiO(100) probed by polarization-dependent spectromicroscopy

Using polarization-dependent x-ray photoemission electron microscopy, we have investigated the surface effects on antiferromagnetic (AFM) domain formation. Depth-resolved information obtained from our study indicates the presence of strain-induced surface AFM domains on some of the cleaved NiO(100) crystals, which are unusually thinner than bulk AFM domain wall widths (similar to 150 nm). Existence of such magnetic skin layer is substantiated by exchange-coupled ferromagnetic Fe domains in Fe/NiO(100), thereby evidencing the influence of this surface AFM domains on interfacial magnetic coupling. Our observations demonstrate a depth evolution of AFM structure in presence of induced surface strain, while the surface symmetry-breaking in absence of induced strain does not modify the bulk AFM domain structure. Realization of such thin surface AFM layer will provide better microscopic understanding of the exchange bias phenomena. (C) 2014 AIP Publishing LLC.

Magnetic structures and magnetic phase transitions in the Mn-doped orthoferrite TbFeO3 studied by neutron powder diffraction

The magnetic structures and the magnetic phase transitions in the Mn-doped orthoferrite TbFeO3 studied using neutron powder diffraction are reported. Magnetic phase transitions are identified at T-N(Fe/Mn) approximate to 295K where a paramagnetic-to-antiferromagnetic transition occurs in the Fe/Mn sublattice, T-SR(Fe/Mn) approximate to 26K where a spin-reorientation transition occurs in the Fe/Mn sublattice and T-N(R) approximate to 2K where Tb-ordering starts to manifest. At 295 K, the magnetic structure of the Fe/Mn sublattice in TbFe0.5Mn0.5O3 belongs to the irreducible representation Gamma(4) (G(x)A(y)F(z) or Pb'n'm). A mixed-domain structure of (Gamma(1) + Gamma(4)) is found at 250K which remains stable down to the spin re-orientation transition at T-SR(Fe/Mn) approximate to 26K. Below 26K and above 250 K, the majority phase (>80%) is that of Gamma(4). Below 10K the high-temperature phase Gamma(4) remains stable till 2K. At 2 K, Tb develops a magnetic moment value of 0.6(2) mu(B)/f.u. and orders long-range in F-z compatible with the Gamma(4) representation. Our study confirms the magnetic phase transitions reported already in a single crystal of TbFe0.5Mn0.5O3 and, in addition, reveals the presence of mixed magnetic domains. The ratio of these magnetic domains as a function of temperature is estimated from Rietveld refinement of neutron diffraction data. Indications of short-range magnetic correlations are present in the low-Q region of the neutron diffraction patterns at T < T-SR(Fe/Mn). These results should motivate further experimental work devoted to measure electric polarization and magnetocapacitance of TbFe0.5Mn0.5O3. (C) 2016 AIP Publishing LLC.

Magnetic structures and magnetic phase transitions in the Mn-doped orthoferrite TbFeO3 studied by neutron powder diffraction

The magnetic structures and the magnetic phase transitions in the Mn-doped orthoferrite TbFeO3 studied using neutron powder diffraction are reported. Magnetic phase transitions are identified at T-N(Fe/Mn) approximate to 295K where a paramagnetic-to-antiferromagnetic transition occurs in the Fe/Mn sublattice, T-SR(Fe/Mn) approximate to 26K where a spin-reorientation transition occurs in the Fe/Mn sublattice and T-N(R) approximate to 2K where Tb-ordering starts to manifest. At 295 K, the magnetic structure of the Fe/Mn sublattice in TbFe0.5Mn0.5O3 belongs to the irreducible representation Gamma(4) (G(x)A(y)F(z) or Pb'n'm). A mixed-domain structure of (Gamma(1) + Gamma(4)) is found at 250K which remains stable down to the spin re-orientation transition at T-SR(Fe/Mn) approximate to 26K. Below 26K and above 250 K, the majority phase (>80%) is that of Gamma(4). Below 10K the high-temperature phase Gamma(4) remains stable till 2K. At 2 K, Tb develops a magnetic moment value of 0.6(2) mu(B)/f.u. and orders long-range in F-z compatible with the Gamma(4) representation. Our study confirms the magnetic phase transitions reported already in a single crystal of TbFe0.5Mn0.5O3 and, in addition, reveals the presence of mixed magnetic domains. The ratio of these magnetic domains as a function of temperature is estimated from Rietveld refinement of neutron diffraction data. Indications of short-range magnetic correlations are present in the low-Q region of the neutron diffraction patterns at T < T-SR(Fe/Mn). These results should motivate further experimental work devoted to measure electric polarization and magnetocapacitance of TbFe0.5Mn0.5O3. (C) 2016 AIP Publishing LLC.